The long-term goal of this research is to provide bright fluorescent sensors for zinc to investigate its neurochemistry. Zinc occurs at high concentrations in vesicles located in presynaptic neurons of the hippocampus and is released into the synaptic cleft in response to a physiological signal. We hypothesize that such zinc release can be used to map neural networks by following the temporal and positional pattern of fluorescence changes that occur following stimulation. Uncontrolled release of neuronal zinc, for example in response to ischemia, leads to Zn-induced death of cortical neurons. The sensors devised here will provide a powerful tool for tracking zinc levels suspected to correlate with such events as well as neurological diseases, including familial amyotrophic lateral sclerosis and Alzheizemer's disease. The proposal focuses on the design and synthesis of three classes of ligands for selective zinc binding, each giving rise to a fluorescent response. The sensors are all derivatives of fluorescein, chosen for its high quantum yield, long wavelength excitation and emission properties, and ability to be manipulated chemically. The first class of ligands improves the brightness of the fluorescence upon Zn2+-binding, which is quenched by photoinduced electron transfer (PET) until zinc binding restores it. This kind of sensor is typified by preliminary work with the "Zinpyr" family of molecules, which contain fluorescein functionalized at the 4' and 5' positions with bis(2-pyridylmethyl)-aminomethyl zinc-binding moieties. A second approach affords ratioable fluorophores by coordination of zinc to the nitrogen atom of a hybrid rhodamine/fluorescein skeleton that we designate as "rhodafluor" ligands. Here, both the unbound and bound sensors fluoresce, but emit at different wavelengths. The third class of molecules to be synthesized and investigated positions the zinc-binding moiety as a spacer between pendant fluorescent donor/acceptor pairs that undergo resonance energy transfer (ET) more efficiently upon zinc binding. All the synthetic routes are modular and convergent, allowing for systematic variation of the Zn2+-binding unit to access a wide range of dissociation constants and solubility properties. The structures, formation constants, rates of formation and dissociation, solubility, solution stability, and fluorescence lifetimes of the zinc complexes of these sensors will be investigated. Their cellular localization will be studied by one- and two-photon microscopic methods. A strategy for attaching the sensors to the extracellular surface of post-synaptic neurons to monitor zinc arrival after synaptic firing will be pursued.